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NEXT GENERATION SEQUENCING

 Name : Alagar . S

Reg no : 2014419002

Branch : M.Tech / Computational Biology

Subject code : BT8001

Subject : Advanced Technologies in Omics Sciences

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DNA SEQUENCING

DNA sequencing is the process of determining the precise orderof  nucleotides within a DNA molecule. It includes any method or technology

that is used to determine the order of the four bases adenine, guanine, cytosine, 

and thymine in a strand of DNA. The advent of rapid DNA sequencing methods

has greatly accelerated biological and medical research and discovery.

NEXT GENERATION SEQUENCING 

 Next-generation sequencing refers to non-Sanger-based high-throughput DNA

sequencing technologies. Millions or billions of DNA strands can be sequenced

in parallel, yielding substantially more throughput and minimizing the need for

the fragment-cloning methods that are often used in Sanger sequencing of

genomes.

ABOUT PACIFIC BIOSCIENCES 

Pacific Biosciences is a biotechnology company founded in 2004 that develops

and manufactures systems for  gene sequencing. They use single molecule real

time sequencing (SMRT) as their platform, based on the properties of  zero-

mode waveguides. The company’s first scientific instrument, called the “PacBio

RS”. A new version of the sequencer called the "PacBio RS II" was then

released. It produces longer sequence reads and offers higher throughput than

the original instrument. In October of 2014 the company released an updated

chemistry with a mean read length of 10,000 to 15,000 bases and longest reads

exceeding 40,000 bases.

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ZERO – MODE WAVEGUIDE

A zero-mode waveguide was developed by Pacific Biosciences, is an optical

waveguide that guides light energy into a volume that is small in all dimensions

compared to the wavelength of the light. The zero-mode waveguide (ZMW) is a

nanophotonic confinement structure that consists of a circular hole in an

aluminum cladding film deposited on a clear silica substrate.[3]

 The ZMW holes

are ~70 nm in diameter and ~100 nm in depth. Due to the behaviour of light

when it travels through a small aperture, the optical field decays exponentially

inside the chamber .[4]

 The observation volume within an illuminated ZMW is

~20 zeptoliters (20 X 10−21

  liters). Within this volume, the activity of DNA

 polymerase incorporating a single nucleotide can be readily detected.

SINGLE MOLECULE REAL TIME SEQUENCING (SMRT)

Single Molecule Real Time Sequencing is the principle technology developed

and used for DNA sequencing by pacific biosciences. Single molecule real time

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sequencing utilizes the zero-mode waveguide.  A single DNA

 polymerase enzyme is affixed at the bottom of a ZMW with a single molecule

of DNA as a template. The ZMW is a structure that creates an illuminated

observation volume that is small enough to observe only a single nucleotide of

DNA (also known as a base) being incorporated by DNA polymerase. Each of

the four DNA bases is attached to one of four different fluorescent dyes. When

a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is

cleaved off and diffuses out of the observation area of the ZMW where its

fluorescence is no longer observable. A detector detects the fluorescent signal of

the nucleotide incorporation, and the base call is made according to the

corresponding fluorescence of the dye.

SMRT technology mimics the natural process of DNA replication, which is a

highly efficient and accurate process. The enzyme responsible for replicating

DNA in nature is called the DNA polymerase. The DNA polymerase attaches

itself to a strand of DNA to be replicated, examines the individual base at the

 point it is attached, and then determines which of four building blocks, or

nucleotides, is required to replicate that individual base. After determining

which nucleotide is required, the polymerase incorporates that nucleotide into

the growing strand that is being produced. After incorporation, the enzyme

advances to the next base to be replicated and the process is repeated. SMRT

technology enables the observation of DNA synthesis as it occurs in real time.

To overcome the challenges inherent in observing an enzyme that is 15

nanometers, or nm, in diameter running in real time, the following key

components are used

  The SMRT Cell

  Phospholinked nucleotides

  The PacBio RS II

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SMRT CELL

SMRT DNA sequencing is performed on SMRT Cells, each patterned with

150,000 zero-mode waveguides or ZMWs. Single polymerases are immobilized

in the ZMWs, which provide the windows to observe DNA sequencing in real-

time. The PacBio RS  System continuously monitors 150,000 ZMWs at a time.

These SMRT Cells are nanofabricated consumable substrates used in

conjunction with the DNA Sequencing Kit for automated processing on the

PacBio RS system.

PHOSPHO LINKED NUCLEOTIDE

For each of the nucleotide bases, there are four corresponding fluorescent dye

molecules that enable the detector to identify the base being incorporated by theDNA polymerase as it performs the DNA synthesis. The fluorescent dye

molecule is attached to the phosphate chain of the nucleotide. When the

nucleotide is incorporated by the DNA polymerase, the fluorescent dye is

cleaved off with the phosphate chain as a part of a natural DNA

synthesis process during which a phospho diester bond is created to elongate the

DNA chain. The cleaved fluorescent dye molecule then diffuses out of thedetection volume so that the fluorescent signal is no longer detected.

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PACBIO RS II

The instrument features high performance optics, automated liquid handling,and an environmental control center, all directed through an intuitive

touchscreen interface. The computational brain responsible for primary data

analysis, called the Blade Center, is also included. This allows for seamless

integration of performance enhancements through chemistry and software

advances.

The instrument contains three primary user access points:

  RS Touch uses a touchscreen interface to start and monitor runs at the

instrument.

  Reagent and Sample Drawer for plate loading.

  SMRT Cell and Tip Drawer for consumables loading.

Throughout the DNA sequencing process, the PacBio RS II uses SMRT

Technology to record light pulses emitted as a byproduct of nucleotide

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incorporation. These signals are delivered in real time to the primary analysis

 pipeline, housed entirely on the Blade Center

Run design and management can be done easily and efficiently through the RS

Remote and RS Touch user interfaces.

RS Touch 

RS Touch, the integrated touchscreen interface, is your primary interaction

 point with the PacBio RS II instrument. It guides you through instrument

loading and sequencing initiation. Intelligent features, including systemwide

 barcode tracking, provide efficient management and high-confidence

 performance. Throughout the single molecule, real-time (SMRT) sequencing

 process, the RS Touch visibly monitors the overall system, including each

SMRT Cell, from preparation through quality assessment. The monitoring

function also displays a real-time sequencing video and expected time to run

completion.

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RS Remote 

The RS Remote is a Windows®-based application that allows you to design and

monitor sequencing runs as well as view primary analysis results, directly from

your desktop. Here, you can define data analysis parameters, including content

and protocol settings, as well as set up automated secondary analysis for custom

data management convenience. The system also has build-in intelligence that

adjusts to provide the user constant feedback on the time and components

needed to complete a run.

Primary Analysis 

Once sequencing is initiated, the system’s computational Blade Center performs

real-time signal processing, base calling and quality assessment. Primary

analysis data, including readlength, distribution, polymerase speed and quality

measurement is streamed directly to the secondary analysis software. This data,

as well as trace and pulse data, are also available through the RS Touch and RS

Remote interfaces for quick assessment of a sequenced SMRT Cell.

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DATA ANALYSIS

SMRT Portal

SMRT Portal is an open-source, browser-based application that allows to create,

submit and monitor secondary analysis jobs as well as view and download the

results. With this tool, you can align reads to a reference sequence or assemble

reads into a de novo genome.

SMRT View

SMRT View is a scalable and extensible application for graphical data

exploration and discovery that allows you to visualize and interact with

secondary analysis sequencing data. A flexible, application-oriented user

interface provides you with a broad range of ways to navigate and explore the

results of your experiments, supporting simple discovery of patterns in your

data. Interactive graphical representations of variants, quality values, and other

metrics, along with rich annotations allow you to rapidly validate your results

 by visualizing genome-scale data sets down to the SNP level. SMRT View,

which includes out-of-the-box support for Sequencing and De Novo workflows,

is the first data visualization application that can visualize kinetics and structure

information unique to PacBio's SMRT technology.

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Data analysis of pacbio RS uses various algorithms for different analytical

 purposes such as

  Hierarchical Genome Assembly Process (HGAP)

  Quiver

  BLASR (Basic Local Alignment with Successive Refinement)

  AHA (A Hybrid Assembler)

  Allora

  Rare and Compound Variants

APPLICATIONS

Applications of Single Molecule Real Time Sequencing involves broad areas of

genomic research such as

  De novo genome sequencing 

  Resequencing 

 

Methylation detection 

  Full isoform sequencing 

  In Vitro Diagnostics. 

REFERENCE

 

http://www.pacificbiosciences.com/   http://www.biomedcentral.com/1471-2164/13/341 

  https://www.youtube.com/playlist?list=PLFAB87D059EC592E9  –  

SMRT Technology Overview

  http://www.nature.com/nmeth/journal/v7/n6/abs/nmeth.1459.html